Oxidized-phospholipids in reconstituted high density lipoprotein particles affect structure and function of recombinant paraoxonase 1

Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, and anti-atherogenic properties. Association of PON1 to HDL particles increases the stability and activity of PON1 and is important for the normal functioning of the enzyme. HDL particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein constituent of HDL that facilitates various biological activities of HDL. In many disease conditions the oxidized phospholipid (Ox-PL) content of HDL is found to be increased and an inverse correlation between the activity of PON1 and oxidation of the HDL is observed. However, the molecular details of the inhibitory action of the Ox-PL-containing HDL on the function of PON1 are not clear yet. In this study we have assembled reconstituted HDL (rHDL) particles with and without Ox-PL and compared their effect on the structure and function of ¹³C-labeled recombinant PON1 (¹³C-rPON1) by employing attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopy and enzymatic assay. Our results show that the presence of the Ox-PL in the rHDL particles alters the structure of rPON1 and decreases its lactonase activity.     

The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.     

Effect in vitro of apolipoprotein A-V on the structure and functions of recombinant high density lipoprotein

The neighboring position of apolipoprotein A-I (apoA-I) and apolipoprotein A-V (apoA-V) gene and the modulation of apoA-V on the concentrations, size and maturation of high density lipoprotein (HDL) may indicate a special relationship between apoA-V and HDL. To assess the effects of apoA-V on HDL structure and related functions in vitro, a series of recombinant HDL (rHDL) were synthesized in vitro with various mass ratios of recombinant apoA-I: apoA-V. An increase in apoA-V in rHDL resulted in enhanced lipid-binding ability, increased phospholipid content and larger particle size. Furthermore, the lipid-free and lipid-bound apoA-V in rHDL showed antioxidant capacity against low density lipoprotein (LDL) in vitro. In THP-1 derived macrophages, apoA-V of rHDL was shown to have no influence on the uptake of oxidized LDL (oxLDL) and intracellular lipid accumulation. Thus, the addition of apoA-V to rHDL resulted in changes in several rHDL properties, including increased lipid-binding ability, phospholipid content, particle size and antioxidant capacity. These alterations may explain the modulation of apoA-V on HDL in vivo and the beneficial functions of apoA-V on atherosclerosis.     

Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

The interplay between size, morphology, stability, and functionality of high-density lipoprotein subclasses

High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDL's in vivo maturation.     

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity

AimsHigh-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised.MethodsReconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL + LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.ResultsrHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.ConclusionsIncreasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics.Non-standard abbreviations and acronyms: AAPH, 2,2′-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.     

Structural and functional properties of apolipoprotein A-I mutants containing disulfide-linked cysteines at positions 124 or 232

Recombinant Cys mutants of apolipoprotein A-I (apoA-I) (A124C and A232C) have been prepared in disulfide-linked forms in order to assess the effects of unnatural covalent constraints on the folding of apoA-I in solution, its ability to bind lipids, form HDL-like particles, activate LCAT, and undergo structural adaptations to changing lipid contents. Both mutants, in dimer form, were shown to fold similarly to plasma apoA-I in solution, but had a slightly decreased alpha-helix content and no evidence of intermonomer interactions. All forms of the mutants bound to and disrupted dimyristoylphosphatidylcholine (DMPC) liposomes with similar kinetics and efficiency to plasma apoA-I, and formed reconstituted HDL (rHDL) particles with palmitoyloleoylphosphatidylcholine (POPC) in high yields at three different ratios of lipid/protein. While the monomeric mutants produced identical rHDL to plasma apoA-I, the disulfide-linked dimers had distinct particle distributions from each other and from native apoA-I. The A124C-dimer formed rHDL with diameters of 86 and 78 A, while the A232C-dimer predominantly formed 96 A rHDL. These particles, and particles containing plasma apoA-I (96 and 78 A), were purified prior to structural and functional analyses. The structural properties of particles with similar diameters were comparable, as were their reactivities with LCAT; however, their ability to undergo structural rearrangements differed. The larger rHDL particles (96 and 86 A) containing native apoA-I or A124C-dimer, rearranged into smaller 78 A particles, while the 96 A particles containing A232C-dimer were resistant to rearrangement and did not form 78 A particles. From the results, it is concluded that synthetic, random disulfide-linked dimers of apoA-I have many properties analogous to those of the naturally occurring Cys mutants, apoA-I-Milano and apoA-I-Paris, which are thought to have antiatherogenic effects in vivo. Also, the results have implications for current models of rHDL structure.     

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.     

Synthesis of reconstituted high density lipoprotein (rHDL) containing apoA-I and apoC-III: the functional role of apoC-III in rHDL

Apolipoprotein (apo) C-III is a marker protein of triacylglycerol (TG)-rich lipoproteins and high-density lipoproteins (HDL), and has been proposed as a risk factor of coronary heart disease. To compare the physiologic role of reconstituted HDL (rHDL) with or without apoC-III, we synthesized rHDL with molar ratios of apoA-I:apoC-III of 1:0, 1:0.5, 1:1, and 1:2. Increasing the apoC-III content in rHDL produced smaller rHDL particles with a lower number of apoA-I molecules. Furthermore, increasing the molar ratio of apoC-III in rHDL enhanced the surfactant-like properties and the ability to lyse dimyristoyl phosphatidylcholine. Furthermore, rHDL containing apoC-III was found to be more resistant to particle rearrangement in the presence of low-density lipoprotein (LDL) than rHDL that contained apoA-I alone. In addition, the lecithin:cholesterol acyltransferase (LCAT) activation ability was reduced as the apoC-III content of the rHDL increased; however, the CE transfer ability was not decreased by the increase of apoC-III. Finally, rHDL containing apoC-III aggravated the production of MDA in cell culture media, which led to increased cellular uptake of LDL. Thus, the addition of apoC-III to rHDL induced changes in the structural and functional properties of the rHDL, especially in particle size and rearrangement and LCAT activation. These alterations may lead to beneficial functions of HDL, which is involved in anti-atherogenic properties in the circulation.     

Cell cholesterol efflux to reconstituted high-density lipoproteins containing the apolipoprotein A-IMilano dimer

The apolipoprotein A-IMilano (apoA-IM) is a molecular variant of apoA-I characterized by the Arg(173)-->Cys substitution, resulting in the formation of homodimers A-IM/A-IM. The introduction of the interchain disulfide bridge in the A-IM dimer limits the apolipoprotein conformational flexibility and restricts HDL particle size heterogeneity, thus possibly affecting HDL function in lipid metabolism and atherosclerosis protection. To investigate whether the structural changes in A-IM/A-IM affect apoA-I capacity for cell cholesterol uptake, we tested the ability of four reconstituted HDL (rHDL), that contained either apoA-I or A-IM/A-IM, to remove cholesterol from Fu5AH hepatoma cells and cholesterol-loaded murine primary macrophages (MPM). As the HDL particle size is known to affect the rHDL capacity for cell cholesterol uptake, the reconstitution conditions were carefully selected to produce two sets of rHDL particles of small and large size (7.8 and 12.5 nm in diameter). The small A-IM/A-IM rHDL were more efficient than the corresponding apoA-I particles as acceptors of membrane cholesterol from Fu5AH cells and MPM, and as inhibitors of cholesterol esterification in MPM. The large rHDL and the lipid-free apolipoproteins displayed instead similar capacities for cell cholesterol efflux. These results suggest that cell cholesterol efflux to rHDL particles of different size occurs through different mechanisms. Large HDL accommodate and retain the cholesterol molecules that have desorbed from the cell membrane into the extracellular fluid, in a process that is less sensitive to protein conformation. Small HDL accelerate the desorption of cholesterol from the cell membrane, in a process that is influenced by the conformation of the proteins on the surface of the acceptor particle. The enhanced efficiency of small A-IM/A-IM rHDL seems related to the peculiar structure of the protein on the rHDL surface, with a hydrophobic C-terminal domain extending out of the rHDL particle, available for anchoring the acceptor to the plasma membrane.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.     

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I

Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and beta-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 A, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed.     

The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.

The details of how high density lipoprotein (HDL) microstructure affects the conformation and net charge of apolipoprotein (apo) A-I in various classes of HDL particles have been investigated in homogeneous recombinant HDL (rHDL) particles containing apoA-I, palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate. Isothermal denaturation with guanidine HCl was used to monitor alpha-helix structural stability, whereas electrokinetic analyses and circular dichroism were used to determine particle charge and apoA-I secondary structure, respectively. Electrokinetic analyses show that at pH 8.6 apoA-I has a net negative charge on discoidal (POPC.apoA-I) particles (-5.2 electronic units/mol of apoA-I) which is significantly greater than that of apoA-I either free in solution or on spherical (POPC.cholesteryl oleate.apoA-I) rHDL (approximately -3.5 electronic units). Raising the POPC content (32-128 mol/ml of apoA-I) of discoidal particles 1) increases the particle major diameter from 9.3 to 12.1 nm, 2) increases the alpha-helix content from 62 to 77%, and 3) stabilizes the helical segments by increasing the free energy of unfolding (delta GD degree) from 1.4 to 3.0 kcal/mol of apoA-I. Raising the POPC content (28-58 mol/mol of apoA-I) of spherical particles 1) increases the particle diameter from 7.4 to 12.6 nm, 2) increases the percent alpha-helix from 62 to 69%, and 3) has no significant effect on delta GD degree (2.2 kcal/mol of apoA-I). This study shows that different HDL subspecies maintain particular apoA-I conformations that confer unique charge and structural characteristics on the particles. It is likely that the charge and conformation of apoA-I are critical molecular properties that modulate the metabolism of HDL particles and influence their role in cholesterol transport.     

High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K(d) < 10(-)(9) M) and slow dissociation rates (t(1/2) > 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K(d) = 1.2 x 10(-)(7) M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by 相似文献   

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10 nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37 °C, but were almost completely denatured around 60 °C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.     

A key point mutation (V156E) affects the structure and functions of human apolipoprotein A-I

A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more alpha-helical and formed a larger complex (110 A) than control apoA-I (97 A). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.     

Serum paraoxonase: effect of the apolipoprotein composition of HDL and the acute phase response

Genetic variations of paraoxonase (PON) correlate with HDL cholesterol and apolipoprotein A-I (apoA-I), suggesting antiatherogenic properties. Atherosclerosis occurs naturally in humans and rabbits but not in mice. We compared variations of PON arylesterase activity (PON AEase, phenylacetate substrate) in humans, rabbits, and mice. In humans and rabbits, >95% of PON AEase is HDL associated. In mice, about 30% of PON AEase is lipid poor. In the absence of apoA-I in mice, total PON AEase is reduced and >60% is lipid poor. PON AEase level and distribution is restored in apoA-I-/- mice injected with adenoviruses encoding human apoA-I and in transgenic mice expressing human apoA-I at a steady-state level. Thus, while apoA-I is not required for the HDL association of PON AEase, induced variations in apoA-I correlate with changes in HDL-associated, but not lipid-poor, PON AEase. PON AEase associates only with apoA-I- or apoE-containing HDL but not VLDL. In the absence of both apoA-I and apoE, PON AEase is all-lipid-poor. PON AEase is displaced from HDL by ultracentrifugation and following incubation with serum amyloid A. Variations in the PON distribution between HDL and lipid-poor fractions may have important consequences in its antioxidant activity and in atherogenesis.     

Apolipoprotein composition and particle size affect HDL degradation by chymase: effect on cellular cholesterol efflux

Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal pre beta-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase. We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.